Cultures preparartion for RNA extraction


1. Cut some mycelial plugs (like 5)  from a 10 -14 days old PDA culture.

2.  Inoculate with the agar plugs  a  flask with 100 ml of Fries medium 48 hours with shaking at 30 C.  I use a 100 mL volume culture in a 250 mL Erlenmyer flask and shake 250rpm .

3. Filtrate culture in funnel with miracloth. Keep mycelium and spores. Trash  agar plugs.

4. Blend the culture filtrated with a 100 ml of new Fries medium. Use a sterile blender.

5. transfer to 1 L sterile  flask . Complete to 400 ml of Fries medium

6. Incubate  24 hs at 30C at 250 rpm (400 ml f Fries media in a 1 L flask)

7.  Filter through a funnel with sterile paper with vacum presure.

8.  Divide into aliquots , cool in liquid N2 for 1 min and freeze at -80C.  I usually use 100 mg in 1.5 ml tubes or 1 gr in a 50 ml tubes aliquots. Also it can be crash in the mortle first and after  that storage.


 Material to be ready before start


Baked:  clean wipe with RNA free and bake 12 hs at 170 C.(second floor)

Mortle and plestle ,  ceramic funnel, spatulas (2)



Sterile Flask 250 ml and 1 L

Sterile Funnel with Miracloth

Sterile whatman paper  (size: ceramic funnel)

 Metal Blender flask ( kennerley lab)

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